Cd34 sc 7324

Flow cytometry assessment was conducted to confirm the mesenchymal origin of cells. Third passage cells were resuspended following digestion with 0.125% trypsin. A minimum of 1 × 10⁵ cells/ml were collected from six-well plates. Rat monoclonal anti-human antibodies were used at a dilution of 1:150 for all cell surface markers. MSCs were incubated with phycoerythrin (PE)-coupled antibodies, CD34 (sc-7324; Santa Cruz) and CD45 (554,878; BD Biosciences), and fluorescein isothiocyanate (FITC)-coupled antibodies, CD44 (550,974; BD Biosciences) and CD105 (BD Biosciences), in the dark at room temperature for 30 min. IgG1-PE and IgG1-FITC were used as isotype controls. Cell data were analyzed using the Paint-A-Gate Pro™ software.

Cell culture materials were purchased from Invitrogen (Carlsbad, CA, USA). Primary antibodies against p-STAT3 (Tyr705) (#9145, 1:1000), STAT3 (#30835, 1:2000), p-p65(Ser536) (#3033, 1:1000), p65 (#8242, 1:2000), and α-SMA (#19245, 1:2000) were purchased from Cell Signaling Technology (Beverly, MA, USA). Primary antibodies against IL-1β (sc-12742, 1:2000), CD34 (sc-7324, 1:2000), VCAM-1 (sc-13160, 1:2000), and EPAS-1 (sc-13596, 1:2000) were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA). Primary antibodies against CD68 (28058-1-AP), ICAM-1 (60299-1-Ig, 1:2000), MCP-1 (66272-1-Ig, 1:2000), Bax (50599-2-Ig, 1:2000), Bcl-2 (12789-1-AP, 1:2000), cleaved-caspase-3 (19677-1-AP, 1:2000), GAPDH (10494-1-AP, 1:2000), FN1 (15613-1-AP, 1:2000), and SOX9 (67439-1-Ig, 1:2000) as well as secondary antibodies (Goat anti-mouse, SA00001-1; Goat anti-rabbit, SA00001-2) were purchased from Proteintech Group (Chicago, IL, USA). Recombinant human interleukin-1 receptor antagonist (IL-1Ra) (SRP3084) was obtained from Sigma (St. Louis, MO, USA), Stattic (inhibitor of STAT3) and Colivelin, (activator of STAT3) were purchased from MedChemExpress (Shanghai, China). Unless otherwise indicated, the remaining reagents used in this study were obtained from Sigma.

ASCs were resuspended following digestion with 0.25% trypsin and washed three times with phosphate-buffered saline (PBS). A minimum of 1 × 10⁵ cells were collected from each sample. Samples were incubated with phycoerythrin (PE)-coupled antibodies against rat CD29 (562,154; BD Biosciences), CD34 (sc-7324; Santa Cruz), CD45 (554,878; BD Biosciences), CD86 (551,396; BD Biosciences), and CD90 (551,401; BD Biosciences), and fluorescein isothiocyanate (FITC)-coupled antibody against rat CD44 (550,974; BD Biosciences) in the dark at room temperature for 30 min. Nonspecific isotype-matched antibodies were used as controls. The samples were subjected to flow cytometry, and data were analyzed using Paint-A-Gate Pro™ software (FACSCalibur™; BD Biosciences).